Artifical nucleases comprising engineered cleavage half-domains

ABSTRACT

Disclosed herein are engineered cleavage half-domains; fusion polypeptides comprising these engineered cleavage half-domains; polynucleotides encoding the engineered cleavage half-domains and fusion proteins; and cells comprising said polynucleotides and/or fusion proteins. Also described are methods of using these polypeptides and polynucleotides, for example for targeted cleavage of a genomic sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/055,525, filed Aug. 6, 2018, which is a continuation of U.S.patent application Ser. No. 15/660,072, filed Jul. 26, 2017, now U.S.Pat. No. 10,066,242, which is a continuation of U.S. patent applicationSer. No. 15/160,571, filed May 20, 2016, now U.S. Pat. No. 9,765,361,which is a continuation of U.S. patent application Ser. No. 14/627,812,filed Feb. 20, 2015, now U.S. Pat. No. 9,376,689, which is acontinuation of U.S. patent application Ser. No. 12/931,660, filed Feb.7, 2011, now U.S. Pat. No. 8,962,281, which claims the benefit of U.S.Provisional Application Nos. 61/337,769, filed Feb. 8, 2010 and61/403,916, filed Sep. 23, 2010, the disclosures of which are herebyincorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 27, 2020, isnamed 8325_0076_07_SL.txt and is 18,726 bytes in size.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the fields of polypeptide and genomeengineering and homologous recombination.

BACKGROUND

Artificial nucleases, such as zinc finger nucleases (fusions of zincfinger domains and cleavage domains) for targeted cleavage of genomicDNA have been described. Such targeted cleavage events can be used, forexample, to induce targeted mutagenesis, induce targeted deletions ofcellular DNA sequences, and facilitate targeted recombination at apredetermined chromosomal locus. See, for example, U.S. PatentPublication Nos. 2003/0232410; 2005/0208489; 2005/0026157; 2005/0064474;2006/0188987; 2006/0063231; and International Publication No. WO07/014275, the disclosures of which are incorporated by reference intheir entireties for all purposes.

To increase specificity, a pair of fusion proteins, each comprising azinc finger binding domain and cleavage half-domain can be used tocleave the target genomic DNA. Because cleavage does not occur unlessthe cleavage half-domains associate to form a functional dimer, thisarrangement increases specificity.

To further decrease off-target cleavage events, engineered cleavagehalf-domains, for example domains that form obligate heterodimers, havealso been developed. See, e.g., U.S. Patent Publication No.2008/0131963. However, there remains a need for additional engineeredcleavage half-domains with increased activity and decreased off-targetcleavage activity.

SUMMARY

The present disclosure provides engineered cleavage half-domains thatexhibit enhanced activity and specificity as compared to wild-typecleavage domains and/or previously described engineered cleavagehalf-domains. Also described are complexes (e.g., heterodimers) andfusion proteins comprising these engineered cleavage half-domains. Thedisclosure also provides methods of using these compositions fortargeted cleavage of cellular chromatin in a region of interest and/orhomologous recombination at a predetermined region of interest in cells.

Thus, in one aspect, described herein is an engineered cleavagehalf-domain comprising two or more mutations as compared to the parentalwild-type cleavage domain from which they are derived. In certainembodiments, the engineered cleavage half-domains are derived from FokIand comprise a mutation in two or more of amino acid residues 418, 432,441, 481, 483, 486, 487, 490, 496, 499, 523, 527, 537, 538 and/or 559,numbered relative to a wild-type FokI cleavage half-domain. In oneembodiment, the engineered cleavage half-domain is derived from awild-type FokI cleavage domain and comprises mutations in amino acidresidues 486, 499 and 496, numbered relative to wild-type FokI. Inanother embodiment, the engineered cleavage half-domain comprisesmutations in amino acid residues 490, 538 and 537, numbered relative towild-type FokI. In another embodiment, the engineered cleavage halfdomains are derived from a wild-type FokI cleavage domain and comprisemutations in the amino acid residues 487, 499 and 496, numbered relativeto wild-type FokI. In one embodiment, the engineered cleavage halfdomains are derived from a wild-type FokI cleavage domain and comprisemutations in the amino acid residues 483, 538 and 537, numbered relativeto wild-type FokI. In still further embodiments, the engineered cleavagehalf-domain comprises mutations in the amino acid residues 490 and 537.

The engineered cleavage half-domains described herein can formheterodimers with wild-type cleavage half-domains and/or with otherengineered cleavage half-domains. In certain embodiments, the engineeredcleavage half-domain comprises mutations at positions 486, 499 and 496(numbered relative to wild-type FokI), for instance mutations thatreplace the wild-type Gln (Q) residue at position 486 with a Glu (E)residue, the wild-type Iso (I) residue at position 499 with a Leu (L)residue and the wild-type Asn (N) residue at position 496 with an Asp(D) or Glu (E) residue (also referred to as a “ELD” and “ELE” domains,respectively). In other embodiments, the engineered cleavage half-domaincomprises mutations at positions 490, 538 and 537 (numbered relative towild-type FokI), for instance mutations that replace the wild-type Glu(E) residue at position 490 with a Lys (K) residue, the wild-type Iso(I) residue at position 538 with a Lys (K) residue, and the wild-typeHis (H) residue at position 537 with a Lys (K) residue or a Arg (R)residue (also referred to as “KKK” and “KKR” domains, respectively). Inother embodiments, the engineered cleavage half-domain comprisesmutations at positions 490 and 537 (numbered relative to wild-typeFokI), for instance mutations that replace the wild-type Glu (E) residueat position 490 with a Lys (K) residue and the wild-type His (H) residueat position 537 with a Lys (K) residue or a Arg (R) residue (alsoreferred to as “KIK” and “KIR” domains, respectively). In still furtherembodiments, the engineered cleavage half-domain comprises mutations atpositions 487 and 496 (numbered relative to wild-type FokI), forinstance mutations that replace the wild-type Arg (R) residue atposition 487 with an Asp (D) residue and the wild-type Asn (N) residueat position 496 with an Asp (D) residue (also referred to as “DD”)and/or mutations at positions 483 and 537 (numbered relative towild-type FokI), for instance, mutations that replace the wild-type Asp(D) residue at position 483 with an Arg (R) residue and the wild-typeHis (H) residue at position 537 with an Arg (R) residue (also referredto as “RR”). In other embodiments, the engineered cleavage half-domaincomprises mutations at positions 487, 499 and 496 (numbered relative towild-type FokI), for instance mutations that replace the wild-type Arg(R) residue at position 487 with an Asp (D) residue and the wild-typeIle (I) residue at position 499 with an Ala (A) and the wild-type Asn(N) residue at position 496 with an Asp (D) residue (also referred to as“DAD”) and/or mutations at positions 483, 538 and 537 (numbered relativeto wild-type FokI), for instance, mutations that replace the wild-typeAsp (D) residue at position 483 with an Arg (R) residue and thewild-type Ile (I) residue at position 538 with a Val (V) residue, andthe wild-type His (H) residue at position 537 with an Arg (R) residue(also referred to as “RVR”).

In another aspect, the engineered cleavage half domains may be furtherengineered to contain mutations in domain of the FokI other than thedimerization domain. For example, mutations at positions 418, 432, 441,481, 523, 527 and 559 have been shown to increase the catalytic activityof a wild-type FokI domain. In particular, the mutations where Pro (P)replaces the wild-type Ser (S) residue at position 418 and where a Glu(E) residue replaces the wild-type Lys (K) residue at position 441(known as “PE”, also known as “Sharkey”) have been shown to enhancecatalytic activity (see Guo, et al., (2010) J. Mol Biol,doi:10.101b/j.jmb.2010.04.060). In another aspect, the mutations wherePro (P) replaces the wild-type Ser (S) at position 418, where Leu (L)replaces the wild-type Phe (F) at position 432, where Glu (E) replacesthe wild-type Lys (K) at position 441, where His (H) replaces thewild-type Gin (Q) at position 481, where Tyr (Y) replaces the wild-typeHis (H) at position 523, where Asp (D) replaces the wild-type Asn (N) atposition 527 and Gin (Q) replaces the wild-type Lys (K) at position 559(known as “Sharkey”, see Guo, et al., ibid). Thus in one embodiment, themutant FokI domain may comprise mutations at positions 418, 441, 486,and 499. In another embodiment, the mutant FokI domain may comprisemutations at positions 418, 441, 490, and 538. In further embodiments,the wild-type FokI domain may be mutated to include mutations atpositions 418, 441, 486, 496 and 499, and/or 418, 441, 490, 537, and538. In other embodiments, the wild-type FokI domain may be mutated atpositions 418, 432, 441, 481, 486, 496, 499 523, 527 and 559 and/orpositions 418, 432, 441, 481, 523, 527, 559, 490, 538 and 537. Inparticular, the mutations may include mutation of the wild-type Gin (Q)at position 486 with Glu (E), mutation of the wild-type lie (I) atposition 499 with a Leu (L), mutation of the wild-type Asn (N) atposition 496 with an Asp (D), mutation of the wild-type Ser (S) atposition 418 with a Pro (P) and mutation of the wild-type Lys (K) atposition 441 with a Glu (E) (also known as “ELD-S” or “ELD Sharkey”)and/or mutation of the wild-type Glu (E) at position 490 with a Lys (K),mutation of the wild-type Ile (I) at position 538 with a Lys (K),mutation of the wild-type His (H) at position 537 with an Lys (K) orArg(R), mutation of the wild-type Ser (S) at position 418 with a Pro (P)and mutation of the wild-type Lys (K) at position 441 with a Glu (E)residue (also known as KKK-S or KKR-S, or KKK-Sharkey or KKR-Sharkey).Further embodiments encompass S418P:F432L:K441E:Q481H:Q486E:N496D:I499L:H523Y:N527D:K559Q, also known as ELD-Sharkey′, andS418P:F432L:K441E:Q481H:E490K:H523Y:N527D:H537K or R:1538K:K559Q, alsoknown as KKK-Sharkey′ or KKR-Sharkey′.

In another aspect, engineered cleavage half domains that displayconditional activity (for example, depending on conditions under whichthe cells are maintained) are provided. In some embodiments, theconditional engineered cleavage half domains display a decrease inactivity under decreased temperature conditions. In some embodiments,the conditional engineered cleavage half domains display a decrease inactivity under increased temperature conditions.

In yet another aspect, engineered cleavage half domains may beincorporated into zinc finger nucleases comprising non-canonicalzinc-coordinating residues (e.g. CCHC rather than the canonical C2H2configuration, see U.S. Patent Publication No. 2003/0108880).

In another aspect, fusion polypeptides comprising a DNA binding domainand an engineered cleavage half-domain as described herein are provided.In certain embodiments, the DNA-binding domain is a zinc finger bindingdomain (e.g., an engineered zinc finger binding domain). In otherembodiments, the DNA-binding domain is a TALE DNA-binding domain.

In another aspect, polynucleotides encoding any of the engineeredcleavage half-domains or fusion proteins as described herein areprovided.

In yet another aspect, cells comprising any of the polypeptides (e.g.,fusion polypeptides) and/or polynucleotides as described herein are alsoprovided. In one embodiment, the cells comprise a pair of fusionpolypeptides, one fusion polypeptide comprising an ELD or ELE cleavagehalf-domain and one fusion polypeptide comprising a KKK or KKR cleavagehalf-domain. In another embodiment, one fusion polypeptide comprises aDAD cleavage half domain while another comprises the RVR fusionpolypeptide. In other embodiments, the paired fusion polypeptidesfurther comprise mutations in other locations of the FokI nucleasedomain. In some embodiments, these catalytic domain mutants are S418Pand K441E, thus these mutant fusion polypeptides comprise the mutantFokI domains listed below:

-   -   (a) EL-S: S418P:K441E:Q486E:I499L    -   (b) KK-S: S418P:K441E:E490K:I538K    -   (c) ELD-S: S418P:K441E:Q486E:N496D:I499L    -   (d) KKK-S: S418P:K441E:E490K:H537K:I538K    -   (e) KKR-S: S418P:K441E:E490K:H537R:I538K    -   (f) DA-S: S418P:K441E:R487D:I499A    -   (g) RV-S: S418P:K441E:D483R:1538V    -   (h) DAD-S: S418P:K441E:R487D:N496D:I499A    -   (i) RVR-S: S418P:K441E:D483R:H537R:1538V    -   (j) DD-S: S418P:K441E:R487D:N496D    -   (k) RR-S: S418P:K441E:D483R:H537R.

In yet another aspect, methods for targeted cleavage of cellularchromatin in a region of interest; methods of causing homologousrecombination to occur in a cell; methods of treating infection; and/ormethods of treating disease are provided. The methods involve cleavingcellular chromatin at a predetermined region of interest in cells byexpressing a pair of fusion polypeptides as described herein (i.e., apair of fusion polypeptides in which one fusion polypeptide comprisesthe engineered cleavage half-domains as described herein).

The engineered cleavage half domains described herein can be used inmethods for targeted cleavage of cellular chromatin in a region ofinterest and/or homologous recombination at a predetermined region ofinterest in cells. Cells include cultured cells, cells in an organismand cells that have been removed from an organism for treatment in caseswhere the cells and/or their descendants will be returned to theorganism after treatment. A region of interest in cellular chromatin canbe, for example, a genomic sequence or portion thereof. Compositionsinclude fusion polypeptides comprising a DNA binding domain (e.g., anengineered zinc finger binding domain or TALE binding domain having anovel specificity) and a cleavage half domain as described.

A fusion protein can be expressed in a cell, e.g., by delivering thefusion protein to the cell or by delivering a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide, if DNA, istranscribed, and an RNA molecule delivered to the cell or a transcriptof a DNA molecule delivered to the cell is translated, to generate thefusion protein. Methods for polynucleotide and polypeptide delivery tocells are presented elsewhere in this disclosure.

Accordingly, in another aspect, a method for cleaving cellular chromatinin a region of interest can comprise (a) selecting a first sequence inthe region of interest; (b) engineering a first DNA binding domain(e.g., zinc finger or TALE DNA binding domain) to bind to the firstsequence; (c) expressing a first fusion protein in the cell, the firstfusion protein comprising the first DNA-binding domain and a firstengineered cleavage half-domain as described herein; and (d) expressinga second fusion protein in the cell, the second fusion proteincomprising a second DNA binding domain and a second cleavage half-domainas described herein, wherein the first fusion protein binds to the firstsequence, and the second fusion protein binds to a second sequencelocated between 2 and 50 nucleotides from the first sequence, therebypositioning the engineered cleavage half-domains such that they form aheterodimer, which heterodimer cleaves cellular chromatin in the regionof interest.

In other embodiments, any of the methods described herein may comprise(a) selecting first and second sequences in a region of interest,wherein the first and second sequences are between 2 and 50 nucleotidesapart; (b) engineering a first DNA binding domain (e.g., zinc finger orTALE DNA binding domain) to bind to the first sequence; (c) engineeringa second zinc finger binding domain to bind to the second sequence; (d)expressing a first fusion protein in the cell, the first fusion proteincomprising the first DNA-binding domain and a first cleavage half-domainas described herein; (e) expressing a second fusion protein in the cell,the second fusion protein comprising the second DNA binding domain(e.g., engineered zinc finger or TALE DNA binding domain) and a secondcleavage half-domain as described herein; wherein the first fusionprotein binds to the first sequence and the second fusion protein bindsto the second sequence, thereby positioning the first and secondengineered cleavage half-domains such that they form a heterodimer whichcleaves the cellular chromatin in the region of interest. In certainembodiments, cellular chromatin is cleaved at one or more sites betweenthe first and second sequences to which the fusion proteins bind.

In further embodiments, a method for cleavage of cellular chromatin in aregion of interest comprises (a) selecting the region of interest; (b)engineering a first DNA binding domain (e.g., zinc finger or TALE DNAbinding domain) to bind to a first sequence in the region of interest;(c) providing a second DNA binding domain (e.g., zinc finger or TALE DNAbinding domain) which binds to a second sequence in the region ofinterest, wherein the second sequence is located between 2 and 50nucleotides from the first sequence; (d) expressing a first fusionprotein in the cell, the first fusion protein comprising the first DNAbinding domain and a first cleavage half-domain as described herein; and(e) expressing a second fusion protein in the cell, the second fusionprotein comprising the second DNA binding domain and a second cleavagehalf domain as described herein; wherein the first fusion protein bindsto the first sequence, and the second fusion protein binds to the secondsequence, thereby positioning the first and 30 second cleavagehalf-domains such that they form a heterodimer and the cellularchromatin is cleaved in the region of interest.

Also provided are methods of altering a region of cellular chromatin,for example to introduce targeted mutations. In certain embodiments,methods of altering cellular chromatin comprise introducing into thecell one or more targeted nucleases to create a double-stranded break incellular chromatin at a predetermined site, and a donor polynucleotide,having homology to the nucleotide sequence of the cellular chromatin inthe region of the break. Cellular DNA repair processes are activated bythe presence of the double-stranded break and the donor polynucleotideis used as a template for repair of the break, resulting in theintroduction of all or part of the nucleotide sequence of the donor intothe cellular chromatin. Thus, a sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide.

Targeted alterations include, but are not limited to, point mutations(i.e., conversion of a single base pair to a different base pair),substitutions (i.e., conversion of a plurality of base pairs to adifferent sequence of identical length), insertions or one or more basepairs, deletions of one or more base pairs and any combination of theaforementioned sequence alterations.

The donor polynucleotide can be DNA or RNA, can be linear or circular,and can be single-stranded or double-stranded. It can be delivered tothe cell as naked nucleic acid, as a complex with one or more deliveryagents (e.g., liposomes, poloxamers) or contained in a viral deliveryvehicle, such as, for example, an adenovirus or an adeno-associatedVirus (AAV). Donor sequences can range in length from 10 to 1,000nucleotides (or any integral value of nucleotides therebetween) orlonger.

In certain embodiments, the frequency of homologous recombination can beenhanced by arresting the cells in the G2 phase of the cell cycle and/orby activating the expression of one or more molecules (protein, RNA)involved in homologous recombination and/or by inhibiting the expressionor activity of proteins involved in non-homologous end-joining.

In any of the methods described herein, the second zinc finger bindingdomain may be engineered to bind to the second sequence.

Furthermore, in any of the methods described herein, the fusion proteinsmay be encoded by a single polynucleotide.

For any of the aforementioned methods, the cellular chromatin can be ina chromosome, episome or organellar genome. Cellular chromatin can bepresent in any type of cell including, but not limited to, prokaryoticand eukaryotic cells, fungal cells, plant cells, animal cells, mammaliancells, primate cells and human cells.

In some aspects, the methods provide for organisms comprising fusionproteins with conditional FokI activity comprising the mutationsdescribed herein. In some embodiments, these organisms are plants. Thesemethods also relate to the tissues of such plants including seeds.

In other embodiments, a method for cleavage of cellular chromatin in twoor more regions of interest is provided. The method comprises (a)selecting the first region of interest; (b) engineering a first DNAbinding domain (e.g., zinc finger or TALE DNA binding domain) to bind toa first sequence in the first region of interest; (c) providing orengineering a second DNA binding domain (e.g., zinc finger or TALE DNAbinding domain) which binds to a second sequence in the first region ofinterest, wherein the second sequence is located between 2 and 50nucleotides from the first sequence; (d) selecting the second region ofinterest; (e) providing or engineering a third DNA binding domain (e.g.,zinc finger or TALE DNA binding domain) to bind to a first sequence inthe second region of interest; (f) providing or engineering a fourth DNAbinding domain (e.g., zinc finger or TALE DNA binding domain) whichbinds to a second sequence in the second region of interest, wherein thesecond sequence is located between 2 and 50 nucleotides from the firstsequence; (g) expressing a first fusion protein in the cell, the firstfusion protein comprising the first DNA binding domain and a firstcleavage half-domain as described herein; and (h) expressing a secondfusion protein in the cell, the second fusion protein comprising thesecond DNA binding domain and a second cleavage half domain as describedherein; wherein the first fusion protein binds to the first sequence,and the second fusion protein binds to the second sequence, therebypositioning the first and second cleavage half-domains such that theyform a heterodimer and the cellular chromatin is cleaved in the firstregion of interest, (i) expression a third fusion protein in the cell,the third fusion protein comprising the third DNA binding domain and athird cleavage half domain as described herein, and (j) expressing afourth fusion protein in the cell, the fourth protein comprising thefourth DNA binding domain and a fourth cleavage half domain as describedherein; wherein the third fusion protein binds to the first sequence inthe second region of interest, and the fourth fusion protein binds tothe second sequence in the second region of interest, thereby positionthe third and fourth cleavage half domains such that they form aheterodimer and the cellular chromatin in cleaved in the second regionof interest.

In addition, in any of the methods described herein, at least one zincfinger binding domain is engineered, for example by design or selectionmethods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B, depict the cleavage activity of zinc finger nucleasescomprising the indicated cleavage domain mutants as described hereinover a range of temperatures of the isolated mutants. FIG. 1A shows theresults when a yeast reporter strain was transformed with the isolatedmutant vectors, divided into three cultures, and incubated at 22° C.,30° C., and 37° C. Following ZFN induction, the activity of the mutantswas determined and reported as a fraction of the wild-type ZFN. FIG. 1Bshows mutant ZFN expression as monitored by western blot using theanti-FLAG antibody. To verify equal protein loading an anti-Histone 3(anti-H3) antibody western blot was performed.

FIGS. 2A and 2B depict the activity of the ZFN variants ELD:KKK andELD:KKR in the 53BPI-specific ZFN background. FIG. 2A depicts theactivity of the 53BP1-specific ZFN variant constructs that werenucleofected in K562 cells. The cells were harvested 3 dayspost-transfection. Two cultures of each combination were assayed (e.g.ELKK 5 and 6). The Cel-1 assay (SurveyorT, Transgenomic) was used todetermine the frequency of ZFN-induced insertions and deletions (%indels), shown in FIG. 2A at the bottom of each lane. ZFN-induced indelsare a consequence of a double strand break (DSB) as a result of ZFNcleavage of the DNA, which is then followed by repair by the cell usingthe Non-Homologous End Joining (NHEJ) process, which can insert ordelete small portions of DNA at the break site during repair. Arrowsindicate expected sizes of bands following Cel-1 cleavage. An aliquot ofthe cells was also cultivated for an additional week to determine thestability of the modified cells in extended cultures (Day 10). FIG. 2Bdepicts ZFN expression as monitored by western blot using the anti-FLAGantibody. As a loading control, anti-NFκB p65 was used.

FIGS. 3A and 3B, show activity of mutants in the KDR-specific ZFNbackground. FIG. 3A depicts Cel-1 activity assays results ofKDR-specific ZFN pairs in K562 cells, monitored at 3 and 20-days postnucleofection. The ZFN pair with indicated FokI mutants used in eachlane is shown above the lane and the activity as detected by the Cel-1assay (as described above for FIG. 2) is shown at the bottom. GFPindicates a negative control. ZFN FokI variants ELD:KKK and ELD:KKR aremore active than the original obligate heterodimeric ZFN (EL:KK). FIG.3B depicts monitoring of ZFN expression and protein loading as describedabove for FIG. 1.

FIG. 4 depicts the activity of ZFN variants ELD:KKK and ELD:KKR in theGR-specific ZFN background, where activity is determined by the Cel-Iassay as described above for FIG. 2. The figure shows the results fromtwo sets of samples for each condition. Lanes 1-14 are one set, andlanes 15-26 are the second set. The novel mutants are more active thanthe original obligate heterodimeric EL:KK at limiting ZFN doses forhighly active ZFNs (compare lane 8 with lanes 9 and 10 and lane 20 withlanes 21 and 22). Decreasing amounts of GR-targeting ZFNs (shown alongthe top of the panel) were nucleofected in K562s, the cells wereharvested 3 days post-transfection, and the Cel-1 assay was used todetermine the frequency of ZFN-induced indels.

FIGS. 5A and 5B depict the activity of mutants in three differentRIPK1-specific ZFN pairs (pairs A, B and C). FIG. 5A depicts Cel-1activity assays (as described above for FIG. 2) for two different ZFNpairs targeting the RIPK1 gene (A and B) that were nucleofected in K562cells. Cells were harvested 3 days post-transfection, and the assayswere used to determine the frequency of ZFN-induced insertions anddeletions (indels). The percent indels are shown at the bottom of eachlane. FIG. 5B shows the results when K562 cells were nucleofected with athird pair of ZFN expression vectors (C) targeting the RIPK1 gene andincubated for 3 days at 37° C. (left panel) or 30° C. (right panel).

FIGS. 6A and 6B show the Cel-1 activity assay results (as describedabove for FIG. 2) of CCR-5-specific ZFN pairs (see, U.S. PatentPublication No. 2008/0159996). FIG. 6A shows the activity of forcedhomodimerization of CCR5 targeting ZFNs after nucleofection of indicatedFokI variants in K562 cells using the Cel-1 assay to determine thefrequency of ZFN-induced indels at the CCR5 heterodimer target, a CCR5-LZFN homodimer (ABLIM2), and a CCR5-R homodimer (PGC) off-target sites.FIG. 6B depicts monitoring of ZFN expression and protein loading asdescribed above for FIG. 1.

FIGS. 7A and 7B shows Cel-1 activity assays results (as described abovefor FIG. 2) of the CCR5 variants described in FIG. 6. FIG. 7A depictsthe Cel-1 activity results using decreasing amounts of CCR5 EL and ELDFokI variants. These constructs were nucleofected in K562 and the Cel-1assay was used to determine the frequency of ZFN-induced indels at theCCR5 heterodimer site. FIG. 7B depicts monitoring of ZFN expression andprotein loading as described above for FIG. 1.

FIGS. 8A and 8B show Cel-1 activity assay results of forcedhomodimerization of mutants in the GR-specific ZFN background. FIG. 8Adepicts the Cel-1 results after forced homodimerization of GR targetingZFNs after nucleofection of indicated FokI variants in K562 cells todetermine the frequency of ZFN-induced indels at the GR heterodimersite. No indels were detectable in this assay in samples other thatwild-type. FIG. 8B depicts monitoring of ZFN expression and proteinloading as described above for FIG. 1.

FIG. 9 depicts flow cytometry data for K562 cells treated with theindicated constructs in the GR targeting ZFN background stained withantibodies against γ-H2AX which targets DSBs. The percent of positivecells is indicated. The percent of DSBs observed for all the FokI mutantpairs was much less than for the wild-type FokI.

FIGS. 10A and 10B depict the activity of the novel ZFN mutants inprimary cells. FIG. 10A shows the Cel-1 activity assay results (asdescribed above for FIG. 2) using decreasing amounts of CCR5-targetingZFNs that were nucleofected in PBMCs. The cells were harvested 3 dayspost-transfection, and the Cel-1 assay was used to determine thefrequency of ZFN-induced indels. FIG. 10B depicts the results in a bargraph with three different ZFN pairs (ZFN A, ZFN B, and ZFN C, seeExample 5) targeting the PD1 gene that were nucleofected in duplicate inPBMCs. The cells were harvested 3 and 10 days post-transfection, and theCel-1 assay was used to determine the frequency of ZFN-induced indels.The graph shows the mean values and error bars from the duplicatetransfections.

FIGS. 11A through 11C depict the activity of the novel ZFN mutants. FIG.11A shows the Cel-1 activity assay results using decreasing amounts ofGR-targeting ZFNs that were nucleofected in PBMCs. The cells wereharvested 3 and 10 days post-transfection, and the Cel-1 assay was usedto determine the frequency of ZFN-induced indels. FIG. 11B depicts a bargraph showing the mean values+/−s.e.m. (standard error of the mean) ofthe relative activities of the indicated ZFNs from six independenttransfections in PBMCs. P-values use the two-sample T-test calculated byMicroCal Origins version 7.5 (OriginLab@), showing the significance ofthe indicated activities with respect to the EL-KK variant. FIG. 11Cdepicts the use of the EL/KK and ELD/KKR GR-specific ZFN pairsnucleofected in K562 cells for promoting targeted integration of a donornucleic acid. The donor in this experiment comprised a novel BamHIrestriction site so that upon targeted integration of the DNA, a PCRamplification product of the targeted area would be able to be cut bythe BamHI restriction enzyme if TI had occurred. The data show that theELD/KKR FokI mutant pair was more efficient at promoting TI than theEL/KK FokI mutant pair.

FIGS. 12A and 12B depict the activity of FokI mutants specific foreither GR or CCR5. FIG. 12A shows the Cel-I activity assay (as describedabove for FIG. 2) results using FokI mutants of GR-targeting ZFNs thatwere nucleofected in K562 cells. See Table 4 for a specific descriptionof the locations of the mutated amino acids. FIG. 12B shows similarresults for CCR5 targeting ZFNs. The results demonstrate that the DA/RVpair is the least active of all FokI mutant pairs tested.

FIG. 13 depicts the activity observed when forced homodimerization ofthe FokI pairs is carried out. Activity is measured by the Cel-1activity assay (as described above for FIG. 2). The ZFNs shown in FIG.13 are specific for GR, and as can be seen from the Figure, the KV FokImutant is the only one in this set to show appreciable homodimerizationactivity.

FIG. 14 depicts the activity as measured by the Cel-1 activity assay(described above for FIG. 2) observed for the enhanced DA/RV pair whichhave been further modified with additional FokI mutations. The Figuredepicts the results for both CCR5-specific and CXCR4-specific ZFN pairswherein the FokI domain has been altered. As can be seen in the results,the additional mutation increases activity of the DA/RV mutantapproximately 2 fold.

FIG. 15 depicts the Cel-I activity of the enhanced DA/RV pair DAD/RVR ina KDR-specific ZFN background. The KDR-specific ZFN pair has weakeractivity to begin with, and the data demonstrate that the DAD/RVR mutantstill does not have detectable activity in this assay and so is similarto the DA/RV pair. In comparison, the ELD/KKR KDR-specific ZFN FokImutant pair shows activity (18-21% indels detected).

FIG. 16 depicts the results following nucleofection of the K562 cellswith two sets of ZFN pairs, specific for CCR5 and CXCR4. The top panelshows the Cel-I assay results (as described above for FIG. 2) for theCCR5 target and the bottom panel shows similar results for the CXCR4target. This experiment was carried out using two parallel incubationconditions, varying only in that one set was maintained only at 37° C.while the other set was held at 30° C. for 3 days. The figure shows thatboth pairs of ZFNs were active simultaneously.

FIG. 17 depicts Cel-I assay cleavage results in K562 cells as above forFIG. 16 except that four potential off-target sites (#3, #5, #7 and #10)were analyzed for cleavage activity. The experiment was as above withtwo incubation conditions. The combination of the ELD/KKR-CCR5 FokImutant pair with the DAD/RVR FokI mutant CXCR4 pair gave undetectableactivity against these 4 off targets in this assay.

FIG. 18 depicts the results of the FokI mutants described in thisinvention in combination with the Sharkey mutant (see Table 4, and Guoet al., ibid) as assayed for activity using the Cel-I assay. The FokImutant pairs tested were either specific for KDR (top panels) or GR(lower panels). The presence of the Sharkey mutation appears to increasethe activity of the other FokI mutants.

FIGS. 19A and 19B depict the activity results of the FokI mutantsdescribed in this invention in combination with the Sharkey mutant (seeTable 4, and Guo et al., ibid) as assayed for activity using the Cel-Iassay. The ZFN FokI mutant pairs tested were specific for GR. Thepresence of the Sharkey mutation appears to increase the activity of theother FokI mutants. FIG. 19A shows the additive results of the FokImutants, and FIG. 19B shows the results of monitoring expression and gelloading as described for Example 1.

FIGS. 20A through 20D depict activity results of various FokImutant+Sharkey FokI mutants are forced to homodimerize as assayed by theCel-I assay. As shown, FokI mutants do not form active homodimercomplexes. FIGS. 20A and 20C show the additive results of the FokImutants, and FIGS. 20B and 20D show the results of monitoring expressionand gel loading as described for Example 1.

FIGS. 21A and 21B depict activity results of D:R and DD:RR FokI mutantsin the context of the indicated ZFNs as assayed by Cel-I assay. FIG. 21Bshows results of monitoring expression and gel loading as described forExample 1.

FIGS. 22A and 22B depict activity results of the various FokI mutants inthe context of the indicated ZFNs at 37° C. (FIG. 22A) or 30° C. (FIG.22B) as assayed by Cel-I assay. The percentage of indels is indicatedbelow the lanes.

DETAILED DESCRIPTION

Disclosed herein are engineered cleavage half-domains and fusionpolypeptides comprising these engineered cleavage half-domains usefulfor targeted cleavage of cellular chromatin and for targeted alterationof a cellular nucleotide sequence, e.g., by targeted cleavage followedby non-homologous end joining or by targeted cleavage followed byhomologous recombination between an exogenous polynucleotide (comprisingone or more regions of homology with the cellular nucleotide sequence)and a genomic sequence.

Exemplary engineered cleavage half-domains are shown in Table 4. Thevariants include mutations such that they form heterodimers with eachother, but not homodimers. This increases the specificity of DNAcleavage and/or increases the concentration of the intended complex (byreducing or eliminating competition from homodimers). When incorporatedinto zinc finger nuclease fusion proteins, these variants induce genemodification at the intended target (both at an endogenous locus andwhen tested using an integrated GFP reporter assay) while significantlyreducing genome wide DNA cleavage as compared to wild-type cleavagehalf-domains.

Thus, the engineered cleavage half-domains described hereinsignificantly impair homodimer function, since forcing two copies of thesame variant to interact reduces or abolishes gene modification. Reducedhomodimer function provides improved ZFN cleavage specificity in vivo,without any decrease in either ZFN expression or the ability tostimulate modification of the desired target site.

In addition, disclosed herein are engineered cleavage half-domains withconditional activity. These conditional mutants can act either ashomodimers or heterodimers, depending on the design. In certainembodiments, conditional activity refers to a change in cleavageactivity based on temperature. Thus, these conditional mutants can beused in the development of cell lines or whole organisms such as plantswherein cleavage activity can be induced by the investigator at certaintemperatures while being held in abeyance at other temperatures.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence. Non-limiting examples of methods forengineering zinc finger proteins are design and selection. A designedzinc finger protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP designs and binding data. See, for example,U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see alsoInternational Patent Publication Nos. WO 98/53058; WO 98/53059; WO98/53060; WO 02/016536; and WO 03/016496.

A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, interaction trap or hybrid selection. See e.g., U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; andInternational Patent Publication Nos. WO 95/19431; WO 96/06166; WO98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; and WO02/099084.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination therebetween, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. Typically thepercent identities between sequences are at least 70-75%, preferably80-82%, more preferably 85-90%, even more preferably 92%, still morepreferably 95%, and most preferably 98% sequence identity.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acid, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain).

A “conditional mutation” is a mutation that has wild-type cleavageactivity under certain permissive environmental conditions and a mutantcleavage activity under certain restrictive conditions. Conditionalmutations may be cold sensitive, where the mutation results in analtered cleavage activity at cooler temperatures, but upon exposure towarmer temperatures, the cleavage activity returns more or less towild-type. Conversely, conditional mutations may be heat sensitive(often termed “thermosensitive”) where the wild type cleavage activityis seen at cooler temperatures but becomes altered upon exposure towarmer temperatures. Altered cleavage activity may be manifested aseither increased or decreased activity.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “accessible region” is a site in cellular chromatin in which a targetsite present in the nucleic acid can be bound by an exogenous moleculewhich recognizes the target site. Without wishing to be bound by anyparticular theory, it is believed that an accessible region is one thatis not packaged into a nucleosomal structure. The distinct structure ofan accessible region can often be detected by its sensitivity tochemical and enzymatic probes, for example, nucleases.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular, and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to a cleavage domain, the ZFP DNA-bindingdomain and the cleavage domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the cleavage domain isable to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain oneore more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA-binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility-shift, orimmunoprecipitation assays. DNA cleavage can be assayed by gelelectrophoresis. See Ausubel et al., supra. The ability of a protein tointeract with another protein can be determined, for example, byco-immunoprecipitation, two-hybrid assays or complementation, bothgenetic and biochemical. See, for example, Fields, et al., (1989) Nature340:245-246; U.S. Pat. No. 5,585,245; and International PatentPublication No. WO 98/44350.

Engineered Cleavage Half-domains

Engineered cleavage half-domains (also referred to as dimerizationdomain mutants) that minimize or prevent homodimerization are describedfor example in U.S. Patent Publication Nos. 2005/0064474; 2006/0188987;and 2008/0131962, incorporated by reference in their entireties herein.Amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490,491, 496, 498, 499, 500, 531, 534, 537, and 538 of FokI are all targetsfor influencing dimerization of the FokI cleavage half-domains.Numbering of amino acid residues in the FokI protein is according toWah, et al., (1998) Proc Nat'l Acad Sci USA 95:10564-10569 (SEQ IDNO:57).

Described herein are engineered cleavage half-domains of FokI thatexhibit increased activity and specificity as compared to previouslydescribed engineered FokI cleavage domains and/or wild-type cleavagedomains. Exemplary mutant cleavage half-domains are shown in Table 3.Exemplary engineered cleavage domains are shown in Table 4. In certainembodiments, the cleavage half-domain includes mutations at least threeamino acid residues at positions, as compared to wild-type. For example,in certain embodiments, the cleavage half-domain includes mutations atpositions 486, 499 and 496. In other embodiments, the cleavagehalf-domain comprises mutations at positions 490, 538 and 537.

In one embodiment, the mutation at 490 replaces Glu (E) with Lys (K);the mutation at 538 replaces lie (1) with Lys (K); the mutation atposition 537 replaces His (H) with Lys (K) or Arg (R); the mutation at486 replaced Gin (Q) with Glu (E); the mutation at position 499 replacesIle (I) with Leucine (L); and the mutation at 496 replaces Asn (N) withAsp (D) or Glu (E). Specifically, the engineered cleavage half-domainsdescribed herein were prepared by mutating positions 490 (E→K), 538(I→K), and 537 (H→K or H→R) in one cleavage half-domain to produceengineered cleavage half-domains designated “E490K:I538K:H537K” (KKK) or“E490K:I538K:H537R” (KKR) and by mutating positions 486 (Q→E), 499 (I→L)and 496 (N→D or N→E) in another cleavage half-domain to produceengineered cleavage half-domains designated “Q486E:I499L:N496E” (ELE) or“Q486E:I499L:N496D” (ELD). The engineered cleavage half-domainsdescribed herein form obligate heterodimer mutants in which aberrantcleavage is minimized or abolished, but activity as compared towild-type is maintained. See Examples.

In other embodiments, the mutation at position 487 replaces Arg (R) withAsp (D) and the Asn (N) at position 496 is replaced with Asp (D) (toproduce R487D:N496D or “DD”) in one cleavage half-domain and by mutationof the wild-type Asp (D) at position 483 to a Arg (R) and mutation ofthe wild-type His (H) as position 537 with Arg (R) (to produceD483R:H537R or “RR”) in the other cleavage half-domain. In still otherembodiments, the mutation at 487 replaces Arg (R) with Asp (D); themutation at position 499 replaces lie (1) with Ala (A) and at position496, the Asn (N) is replaced with Asp (D) (to produce“R487D:N496D:I499A” in one cleavage half domain) and by mutation atposition 483 (D->R), 538 (I->V) and 537 (H->R) to produce“D483R:H537R:I538V:” at the other cleavage half domain (or DAD and RVR).

In other embodiments, mutations are made in other domains, for exampleat positions 418, 432, 441, 481, 523, 527 and/or 559. In certainembodiments, mutations made at positions 418 and 441, for example areplacement of the wild-type Ser (S) at position 418 with a Pro (P)residue and replacement of the wild-type Lys (K) at position 441 with aGlu (E), known as “S418P:K441E” or “Sharkey”, or where Pro (P) replacesSer (S) at 418, Leu (L) replaces Phe (F) at 432, Glu (E) replaces Lys(K) at 441, His (H) replaces Gln (Q) at 481, Tyr (Y) replaces His (H) at523, Asp (D) replaces Asn (N) at 527 and Gin (Q) replaces Lys (K) atposition 539, known as S418P:F432L:K441E:Q481H:H523Y:N527D:K539Q orSharkey′. These mutations may be combined in any way with the domainslisted above to produce, for example, the following FokI mutants:

-   -   (a) EL-S: S418P:K441E:Q486E:I499L    -   (b) KK-S: S418P:K441E:E490K:I538K    -   (c) ELD-S: S418P:K441E:Q486E:N496D:I499L    -   (d) KKK-S: 5418P:K441E:E490K:H537K:I538K    -   (e) KKR-S: S418P:K441E:E490K:H537R:I538K    -   (f) DA-S: S418P:K441E:R487D:I499A    -   (g) RV-S: S418P:K441E:D483R:I538V    -   (h) DAD-S: S418P:K441E:R487D:N496D:I499A    -   (i) RVR-S: S418P:K441E:D483R:H537R:1538V    -   (j) DD-S: S418P:K441E:R487D:N496D    -   (k) RR-S: S418P:K441E:D483R:H537R.

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (FokI) as described in Example 5 of U.S.Patent Publication No. 2005/0064474 and Examples 5 and 38 ofInternational Patent Publication WO 07/014275.

Fusion Proteins

The engineered cleavage half-domains described herein are advantageouslyused in fusion proteins with DNA binding proteins to specifically targetsites for cleavage in any cell.

In certain embodiments, the DNA binding protein comprises a zinc fingerprotein (ZFP). Selection of target sites; ZFPs and methods for designand construction of fusion proteins (and polynucleotides encoding same)are known to those of skill in the art and described in detail in U.S.Patent Publication Nos. 2005/0064474 and 2006/0188987, incorporated byreference in their entireties herein.

In some embodiments, the DNA binding domain is an engineered domain froma TAL effector derived from the plant pathogen Xanthomonas (see, Miller,et al., (2010) Nature Biotechnology, December 22 [Epub ahead of print];Boch, et al., (2009) Science 29 Oct. 2009 (10.1126/science. 117881) andMoscou and Bogdanove, (2009) Science 29 Oct. 2009(10.1126/science.1178817); see, also, U.S. Patent Publication No.2011/0301073, the disclosure of which is hereby incorporated byreference in its entirety. In some embodiments, the TALE DNA bindingdomain is fused to a FokI cleavage as described, resulting in aTALE-nuclease (TALEN).

The nucleases (e.g., ZFNs) described herein may be delivered to a targetcell by any suitable means. Methods of delivering proteins comprisingzinc fingers are described, for example, in U.S. Pat. Nos. 6,453,242;6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978;6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of allof which are incorporated by reference herein in their entireties.

Fusion proteins (nucleases) as described herein may also be deliveredusing vectors containing sequences encoding one or more of the nucleases(e.g., ZFNs or TALENS). Any vector systems may be used including, butnot limited to, plasmid vectors, retroviral vectors, lentiviral vectors,adenovirus vectors, poxvirus vectors; herpesvirus vectors andadeno-associated virus vectors, etc. See, also, U.S. Pat. Nos.6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and7,163,824, incorporated by reference herein in their entireties.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding nucleases (e.g., ZFNs or TALENS)comprising engineered cleavage domains in cells (e.g., mammalian cells)and target tissues. Such methods can also be used to administer suchnucleic acids to cells in vitro. In certain embodiments, nucleic acidsencoding the one or more nucleases are administered for in vivo or exvivo gene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome or poloxamer. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson, Science 256:808-813 (1992); Nabel and Fegner,TIBTECH 11:211-217 (1993); Mitani and Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer and Perricaudet,British Medical Bulletin 51(1):31-44 (1995); Haddada, et al., in CurrentTopics in Microbiology and Immunology Doerfler and Bôhm (eds.) (1995);and Yu, et al., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.) and BTX Molecular Delivery Systems (Holliston, Mass.).

Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, International PatentPublication Nos. WO 91/17424, WO 91/16024. Delivery can be to cells (exvivo administration) or target tissues (in vive administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese, etal., Cancer Gene Ther. 2:291-297 (1995); Behr, et al., BioconjugateChem. 5:382-389 (1994); Remy, et al., Bioconjugate Chem. 5:647-654(1994); Gao, et al., Gene Therapy 2:710-722 (1995); Ahmad, et al., 15Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183; 4,217,344;4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding nucleases (e.g., ZFNs or TALENS) comprising engineeredcleavage half-domains as described herein take advantage of highlyevolved processes for targeting a virus to specific cells in the bodyand trafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of nucleases asdescribed herein include, but are not limited to, retroviral,lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplexvirus vectors for gene transfer. Integration in the host genome ispossible with the retrovirus, lentivirus, and adeno-associated virusgene transfer methods, often resulting in long term expression of theinserted transgene. Additionally, high transduction efficiencies havebeen observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher, et al., J. Virol.66:2731-2739 (1992); Johann, et al., J. Virol. 66:1635-1640 (1992);Sommerfelt, et al., Virol. 176:58-59 (1990); Wilson, et al., J. Virol.63:2374-2378 (1989); Miller, et al., J. Virol. 65:2220-2224 (1991);International Patent Publication No. WO 1994/026877).

In applications in which transient expression of a ZFP fusion protein ispreferred, adenoviral based systems can be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand high levels of expression have been obtained. This vector can beproduced in large quantities in a relatively simple system.Adeno-associated virus (“AAV”) vectors are also used to transduce cellswith target nucleic acids, e.g., in the in vitro production of nucleicacids and peptides, and for in vivo and ex vivo gene therapy procedures(see, e.g., West, et al., Virology 160:38-47 (1987); U.S. Pat. No.4,797,368; International Patent Publication No. WO 93/24641; Kotin,Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351(1994). Construction of recombinant AAV vectors is described in a numberof publications, including U.S. Pat. No. 5,173,414; Tratschin, et al.,Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al, Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar, et al., Blood 85:3048-305 (1995); Kohn, etal., Nat. Med. 1:1017-102 (1995); Malech, et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese, et al., Science 270:475-480 (1995)).Transduction efficiencies of 50% or greater have been observed for MFG-Spackaged vectors. (Ellem, et al., Immunol Immunother. 44(1):10-20(1997); Dranoff, et al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner, et al., Lancet 351:9117 1702-3 (1998), Kearns et al., GeneTher. 9:748-55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman, et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker, et al.,Infection 24:1 5-10 (1996); Sterman, et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh, et al., Hum. Gene Ther. 2:205-18 (1995);Alvarez, et al., Hum. Gene Ther. 5:597-613 (1997); Topf, et al., GeneTher. 5:507-513 (1998); Sterman, et al., Hum. Gene Ther. 7:1083-1089(1998).

In certain embodiments, the vector is an adenovirus vector. Thus,described herein are adenovirus (Ad) vectors for introducingheterologous sequences (e.g., zinc finger or TALE nucleases (ZFNs orTALENs)) into cells.

Non-limiting examples of Ad vectors that can be used in the presentapplication include recombinant (such as E1-deleted), conditionallyreplication competent (such as oncolytic) and/or replication competentAd vectors derived from human or non-human serotypes (e.g., Ad5, Ad11,Ad35, or porcine adenovirus-3); and/or chimeric Ad vectors (such asAd5/35) or tropism-altered Ad vectors with engineered fiber (e.g., knobor shaft) proteins (such as peptide insertions within the HI loop of theknob protein). Also useful are “gutless” Ad vectors, e.g., an Ad vectorin which all adenovirus genes have been removed, to reduceimmunogenicity and to increase the size of the DNA payload. This allows,for example, simultaneous delivery of sequences encoding ZFNs and adonor sequence. Such gutless vectors are especially useful when thedonor sequences include large transgenes to be integrated via targetedintegration.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer, and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in cells that provide one or more of thedeleted gene functions in trans. For example, human 293 cells supply E1function. Ad vectors can transduce multiple types of tissues in vivo,including non-dividing, differentiated cells such as those found inliver, kidney and muscle. Conventional Ad vectors have a large carryingcapacity. An example of the use of an Ad vector in a clinical trialinvolved polynucleotide therapy for antitumor immunization withintramuscular injection (Sterman, et al., Hum. Gene Ther. 7:1083-1089(1998)).

Additional examples of the use of adenovirus vectors for gene transferin clinical trials include Rosenecker, et al., Infection 24:1 5-10(1996); Welsh, et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez, et al.,Hum. Gene Ther. 5:597-613 (1997); Topf, et al., Gene Ther. 5:507-513(1998).

In certain embodiments, the Ad vector is a chimeric adenovirus vector,containing sequences from two or more different adenovirus genomes. Forexample, the Ad vector can be an Ad5/35 vector. Ad5/35 is created byreplacing one or more of the fiber protein genes (knob, shaft, tail,penton) of Ad5 with the corresponding fiber protein gene from a B groupadenovirus such as, for example, Ad35. The Ad5/35 vector andcharacteristics of this vector are described, for example, in Ni, etal., (2005) Hum Gene Ther 16:664-677; Nilsson, et al., (2004) Mol Ther9:377-388; Nilsson, et al., (2004) J Gene Med 6:631-641; Schroers, etal., (2004) Exp Hematol 32:536-546; Seshidhar, et al., (2003) Virology311:384-393; Shayakhmetov, et al., (2000) J Virol 74:2567-2583; andSova, et al., (2004), Mol Ther 9:496-509.

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han, et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a ZFN orTALEN nucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney, et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba, et al., J. Exp.Med. 176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+(T cells), CD45+ (panB cells), GR-1 (granulocytes),and lad (differentiated antigen presenting cells) (see Inaba, et al., J.Exp. Med. 176:1693-1702 (1992)). In some instances, the stem cells areinduced pluripotent stem cells (iPSC).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP or TALE nucleic acids can also be administered directlyto an organism for transduction of cells in vivo. Alternatively, nakedDNA can be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638. Vectors useful forintroduction of transgenes into hematopoietic stem cells, e.g., CD34⁺cells, include adenovirus Type 35.

Vectors suitable for introduction of transgenes into immune cells (e.g.,T-cells) include non-integrating lentivirus vectors. See, for example,Ory, et al., (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull, etal., (1998) J. Virol. 72:8463-8471; Zuffery, et al., (1998) J. Virol.72:9873-9880; Follenzi, et al., (2000) Nature Genetics 25:217-222.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

As noted above, the disclosed methods and compositions can be used inany type of cell including, but not limited to, prokaryotic cells,fungal cells, Archaeal cells, plant cells, insect cells, animal cells,vertebrate cells, mammalian cells and human cells. Suitable cell linesfor protein expression are known to those of skill in the art andinclude, but are not limited to COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44,CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0,SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6,insect cells such as Spodoptera fugiperda (Sf), and fungal cells such asSaccharomyces, Pischia and Schizosaccharomyces. Progeny, variants andderivatives of these cell lines can also be used.

Applications

The disclosed cleavage domains are advantageously used in combinationwith DNA-binding domains such as zinc finger proteins or TAL bindingdomains (resulting in ZFNs or TALENs respectively) to cleave DNA andminimize off-target site cleavage (as compared to DNA-binding domainscomprising wild-type or homodimerizing cleavage domains). Cleavage canbe at one or more region(s) of interest in cellular chromatin (e.g., ata desired or predetermined site in a genome, for example, in a gene,either mutant or wild-type); to replace a genomic sequence (e.g., aregion of interest in cellular chromatin) with a homologousnon-identical sequence (i.e., targeted recombination); to delete agenomic sequence by cleaving DNA at one or more sites in the genome,which cleavage sites are then joined by non-homologous end joining(NHEJ); to screen for cellular factors that facilitate homologousrecombination; and/or to replace a wild-type sequence with a mutantsequence, or to convert one allele to a different allele. Such methodsare described in detail, for example, in U.S. Patent Publication No.2005/0064474; International Patent Publication No. WO 07/014275,incorporated by reference in their entireties herein.

Accordingly, the disclosed engineered cleavage half domains can be usedin any ZFN or TALEN for any method in which specifically targetedcleavage is desirable and/or to replace any genomic sequence with ahomologous, non-identical sequence. For example, a mutant genomicsequence can be replaced by its wild-type counterpart, thereby providingmethods for treatment of e.g., genetic disease, inherited disorders,cancer, and autoimmune disease. In like fashion, one allele of a genecan be replaced by a different allele using the methods of targetedrecombination disclosed herein. Indeed, any pathology dependent upon aparticular genomic sequence, in any fashion, can be corrected oralleviated using the methods and compositions disclosed herein.

Exemplary genetic diseases include, but are not limited to,achondroplasia, achromatopsia, acid maltase deficiency, adenosinedeaminase deficiency (OMIM No. 102700), adrenoleukodystrophy, aicardisyndrome, alpha-1 antitrypsin deficiency, alpha-thalassemia, androgeninsensitivity syndrome, apert syndrome, arrhythmogenic rightventricular, dysplasia, ataxia telangictasia, barth syndrome,beta-thalassemia, blue rubber bleb nevus syndrome, canavan disease,chronic granulomatous diseases (CGD), cri du chat syndrome, cysticfibrosis, dercum's disease, ectodermal dysplasia, fanconi anemia,fibrodysplasia ossificans progressive, fragile X syndrome, galactosemis,Gaucher's disease, generalized gangliosidoses (e.g., GM1),hemochromatosis, the hemoglobin C mutation in the 6^(th) codon ofbeta-globin (HbC), hemophilia, Huntington's disease, Hurler Syndrome,hypophosphatasia, Klinefleter syndrome, Krabbes Disease, Langer-GiedionSyndrome, leukocyte adhesion deficiency (LAD, OMIM No. 116920),leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome,mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetesinsipdius, neurofibromatosis, Neimann-Pick disease, osteogenesisimperfecta, porphyria, Prader-Willi syndrome, progeria, Proteussyndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome,Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachmansyndrome, sickle cell disease (sickle cell anemia), Smith-Magenissyndrome, Stickler syndrome, Tay-Sachs disease, Thrombocytopenia AbsentRadius (TAR) syndrome, Treacher Collins syndrome, trisomy, tuberoussclerosis, Turner's syndrome, urea cycle disorder, von Hippel-Landaudisease, Waardenburg syndrome, Williams syndrome, Wilson's disease,Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome (XLP,OMIM No. 308240).

Additional exemplary diseases that can be treated by targeted DNAcleavage and/or homologous recombination include acquiredimmunodeficiencies, lysosomal storage diseases (e.g., Gaucher's disease,GM1, Fabry disease and Tay-Sachs disease), mucopolysaccahidosis (e.g.Hunter's disease, Hurler's disease), hemoglobinopathies (e.g., sicklecell diseases, HbC, α-thalassemia, β-thalassemia) and hemophilias.

Such methods also allow for treatment of infections (viral or bacterial)in a host (e.g., by blocking expression of viral or bacterial receptors,thereby preventing infection and/or spread in a host organism) to treatgenetic diseases.

Targeted cleavage of infecting or integrated viral genomes can be usedto treat viral infections in a host. Additionally, targeted cleavage ofgenes encoding receptors for viruses can be used to block expression ofsuch receptors, thereby preventing viral infection and/or viral spreadin a host organism. Targeted mutagenesis of genes encoding viralreceptors (e.g., the CCR5 and CXCR4 receptors for HIV) can be used torender the receptors unable to bind to virus, thereby preventing newinfection and blocking the spread of existing infections. See, U.S.Patent Application No. 2008/015996. Non-limiting examples of viruses orviral receptors that may be targeted include herpes simplex virus (HSV),such as HSV-1 and HSV-2, varicella zoster virus (VZV), Epstein-Barrvirus (EBV) and cytomegalovirus (CMV), HHV6 and HHV7. The hepatitisfamily of viruses includes hepatitis A virus (HAV), hepatitis B virus(HBV), hepatitis C virus (HCV), the delta hepatitis virus (HDV),hepatitis E virus (HEV) and hepatitis G virus (HGV). Other viruses ortheir receptors may be targeted, including, but not limited to,Picornaviridae (e.g., polioviruses, etc.); Caliciviridae; Togaviridae(e.g., rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae;Reoviridae; Bimaviridae; Rhabodoviridae (e.g., rabies virus, etc.);Filoviridae; Paramyxoviridae (e.g., mumps virus, measles virus,respiratory syncytial virus, etc.); Orthomyxoviridae (e.g., influenzavirus types A, B and C, etc.); Bunyaviridae; Arenaviridae; Retroviradae;lentiviruses (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV,ARV, hTLR, etc.) HIV-II); simian immunodeficiency virus (SIV), humanpapillomavirus (HPV), influenza virus and the tick-borne encephalitisviruses. See, e.g. Virology, 3rd Edition (W. K. Joklik ed. 1988);Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds.1991), for a description of these and other viruses. Receptors for HIV,for example, include CCR-5 and CXCR-4.

Thus, heterodimeric cleavage domain variants as described herein providebroad utility for improving ZFN specificity in gene modificationapplications. These variant cleavage domains may be readily incorporatedinto any existing ZFN by either site directed mutagenesis or subcloningto improve the in vivo specificity of any ZFN dimers.

As noted above, the compositions and methods described herein can beused for gene modification, gene correction, and gene disruption.Non-limiting examples of gene modification includes homology directedrepair (HDR)-based targeted integration; HDR-based gene correction;HDR-based gene modification; HDR-based gene disruption; NHEJ-based genedisruption and/or combinations of HDR, NHEJ, and/or single strandannealing (SSA). Single-Strand Annealing (SSA) refers to the repair of adouble strand break between two repeated sequences that occur in thesame orientation by resection of the DSB by 5′-3′ exonucleases to exposethe 2 complementary regions. The single-strands encoding the 2 directrepeats then anneal to each other, and the annealed intermediate can beprocessed such that the single-stranded tails (the portion of thesingle-stranded DNA that is not annealed to any sequence) are bedigested away, the gaps filled in by DNA Polymerase, and the DNA endsrejoined. This results in the deletion of sequences located between thedirect repeats.

Compositions comprising cleavage domains (e.g., ZFNs) and methodsdescribed herein can also be used in the treatment of various geneticdiseases and/or infectious diseases.

The compositions and methods can also be applied to stem cell basedtherapies, including but not limited to: correction of somatic cellmutations by short patch gene conversion or targeted integration formonogenic gene therapy; disruption of dominant negative alleles;disruption of genes required for the entry or productive infection ofpathogens into cells; enhanced tissue engineering, for example, bymodifying gene activity to promote the differentiation or formation offunctional tissues; and/or disrupting gene activity to promote thedifferentiation or formation of functional tissues; blocking or inducingdifferentiation, for example, by disrupting genes that blockdifferentiation to promote stem cells to differentiate down a specificlineage pathway, targeted insertion of a gene or siRNA expressioncassette that can stimulate stem cell differentiation, targetedinsertion of a gene or siRNA expression cassette that can block stemcell differentiation and allow better expansion and maintenance ofpluripotency, and/or targeted insertion of a reporter gene in frame withan endogenous gene that is a marker of pluripotency or differentiationstate that would allow an easy marker to score differentiation state ofstem cells and how changes in media, cytokines, growth conditions,expression of genes, expression of siRNA, shRNA or miRNA molecules,exposure to antibodies to cell surface markers, or drugs alter thisstate; somatic cell nuclear transfer, for example, a patient's ownsomatic cells can be isolated, the intended target gene modified in theappropriate manner, cell clones generated (and quality controlled toensure genome safety), and the nuclei from these cells isolated andtransferred into unfertilized eggs to generate patient-specific hEScells that could be directly injected or differentiated beforeengrafting into the patient, thereby reducing or eliminating tissuerejection; universal stem cells by knocking out MHC receptors (e.g., togenerate cells of diminished or altogether abolished immunologicalidentity). Cell types for this procedure include but are not limited to,T-cells, B cells, hematopoietic stem cells, and embryonic stem cells.Additionally, induced pluripotent stem cells (iPSC) may be used whichwould also be generated from a patient's own somatic cells. Therefore,these stem cells or their derivatives (differentiated cell types ortissues) could be potentially engrafted into any person regardless oftheir origin or histocompatibility.

The compositions and methods can also be used for somatic cell therapy(e.g., autologous cell therapy and/or universal T-cell by knocking outMHC or viral receptors, see above), thereby allowing production ofstocks of T-cells that have been modified to enhance their biologicalproperties. Such cells can be infused into a variety of patientsindependent of the donor source of the T-cells and theirhistocompatibility to the recipient.

In addition to therapeutic applications, the increased specificityprovided by the variants described herein when used in ZFNs can be usedfor crop engineering, cell line engineering and the construction ofdisease models. The obligate heterodimer cleavage half-domains provide astraightforward means for improving ZFN properties, especially whenhomodimer activity limits efficacy.

The engineered cleavage half domains described can also be used in genemodification protocols requiring simultaneous cleavage at multipletargets either to delete the intervening region or to alter two specificloci at once. Cleavage at two targets would require cellular expressionof four ZFNs, which could yield potentially ten different active ZFNcombinations. For such applications, substitution of these novelvariants for the wild-type nuclease domain would eliminate the activityof the undesired combinations and reduce chances of off-target cleavage.If cleavage at a certain desired DNA target requires the activity of theZFN pair A+B, and simultaneous cleavage at a second desired DNA targetrequires the activity of the ZFN pair X+Y, then use of the mutationsdescribed herein can prevent the pairings of A with A, A with X, A withY and so on. Thus, these FokI mutations decrease non-specific cleavageactivity as a result of “illegitimate” pair formation and allow thegeneration of more efficient orthogonal mutant pairs of ZFNs (seeco-owned patent U.S. Patent Publication Nos. 2008/0131962 and2009/0305346).

In addition to the applications described for the engineered cleavagehalf domains, there are also numerous applications for the conditionalmutations described herein. The identified cold-sensitive mutations canbe used to create transgenic organisms carrying an integrated copy ofthe nucleic acid encoding the mutations. Plants carrying such mutationswould display the mutant phenotype such that the cleavage activity wouldbe quiescent at cooler temperatures. Upon a shift to highertemperatures, the fusion would display active cleavage activity. Thesemutant organisms could be used to create lines for breeding purposeswhere lines containing the cold sensitive mutation could be crossed tolines carrying a certain target such that when the progeny of the crosswere shifted to higher temperatures, cleavage of the target would occur.This would increase the efficiency of such processes because it wouldreduce the number of plant transformations with either donor or fusionprotein that would be required to achieve a desired result. The sametype of scenario can also be envisioned for thermo sensitive conditionalmutants.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entireties.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity and understanding,it will be apparent to those of skill in the art that various changesand modifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing disclosure andfollowing examples should not be construed as limiting.

Examples Example 1: Preparation of ZFNs

ZFNs targeted to CCR5, 53BP1, GR, KDR, RIPK1, CXCR4 and PD-1 weredesigned and incorporated into plasmids vectors essentially as describedin Urnov, et al., (2005) Nature 435(7042):646-651, Perez, et al., (2008)Nature Biotechnology 26(7): 808-816, and U.S. Patent Publication No.2008/0131962 or were obtained from Sigma Aldrich. These ZFNs wereconstructed and tested by ELISA and the Surveyor™ (Transgenomics) Cel-1assay (“Cel-1”) as described in Miller, et al., (2007) Nat. Biotechnol.25:778-785 and U.S. Patent Publication No. 2005/0064474 andInternational Patent Publication No. WO 2005/014791. In addition, seeU.S. Patent Publication No. 2008/0188000 for ZFNs targeted to GR, andU.S. Provisional Application No. 61/281,432 relating to ZFNs targeted toPD-1, U.S. Patent Publication No. 2008/0159996 relating to CCR5-specificZFNs and U.S. Pat. No. 8,871,905 relating to CXCR4-specific ZFNs.

Specific examples of ZFPs targeted to RIPK1, KDR and 53BP1 are disclosedin Table 1. The first column in this table is an internal reference name(number) for a ZFP. “F” refers to the finger and the number following“F” refers to which zinc finger (e.g., “F1” refers to finger 1). Table 2lists target binding sites on the target genes. Nucleotides in thetarget site that are contacted by the ZFP recognition helices areindicated in uppercase letters; non-contacted nucleotides indicated inlowercase.

TABLE 1 ZFP designs for 53BP1, KDR and RIPK1 SBS # (tar- Design get) F1F2 F3 F4 F5 F6 18346 RSDHLST TSANLSR RSDNLSE TSGSLTR N/A N/A (53BP1)(SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID  NO: 1) NO: 2) NO: 3) NO: 4) 18347QSGALAR RSDNLTR QSGNLAR QSGNLAR QSGHL QSSDL (53BP1) (SEQ ID  (SEQ ID (SEQ ID  (SEQ ID  QR   RR NO: 5) NO: 6) NO: 7) NO: 7) (SEQ (SEQ ID IDNO:  NO:  8) 9) 19135 RSDTLSE TSGSLTR RSDNLSR QNAHRTT QSSNL RSDDL (KDR)(SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID  AR    TR   NO: 10) NO: 4) NO: 11)NO: 12) (SEQ  (SEQ  ID ID NO: NO: 13) 14) 19136 DRSHLSR QSGNLAR DNPNLNRRSDDLSR RSDNL RNAHR (KDR) (SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID  SE   IN  NO: 15) NO: 7) NO: 16) NO: 17) (SEQ  (SEQ  ID ID NO: NO: 3) 18) 19119RSANLTR RSDNLSE ASKTRKN DRSNLSR TSANL N/A (RIPK1) (SEQ ID  (SEQ ID (SEQ ID  (SEQ ID  SR   NO: 19) NO: 3) NO: 20) NO: 21) (SEQ  ID NO: 2)19120 QSGALAR QSGNLAR RSDHLSA QSGHLSR N/A N/A (RIPK1) (SEQ ID  (SEQ ID (SEQ ID  (SEQ ID  NO: 5) NO: 7) NO: 22) NO: 23) 19123 TSGSLSR QSSDLRRRSDTLSA DNSNRIK RSAAL QSGDL (RIPK1) (SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID SR   TR   NO: 24) NO: 9) NO: 25) NO: 26) (SEQ  (SEQ  ID ID NO: NO: 27)28) 19124 QSGHLSR RSDSLSA DRSNLTR RSDNLSQ ASNDR N/A (RIPK1) (SEQ ID (SEQ ID  (SEQ ID  (SEQ ID  KK   NO: 23) NO: 29) NO: 30) NO: 31) (SEQ  IDNO: 32) 19121 RSDNLSR DSSTRKK RSDNLSV DRSHLAR QSGHL N/A (RIPK1) (SEQ ID (SEQ ID  (SEQ ID  (SEQ ID  SR   NO: 11) NO: 33) NO: 34) NO: 35) (SEQ  IDNO: 23) 19122 QRSNLVR QSSDLTR GNVDLIE RSSNLSR RSDSL TNHNR (RIPK1)(SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID  SV   KT   NO: 36) NO: 37) NO: 38)NO: 39) (SEQ  (SEQ  ID ID NO: NO: 40) 41)

TABLE 2 ZFN Target sites SBS# (target) Target site 18346ttGTTCAGGATTGGacacaacatcctag_ (53BP1) (SEQ ID NO: 42) 18347caGCTGGAGAAGAAcGAGGAGacggtaa_ (53BP1) (SEQ ID NO: 43) 19135ctGCGGATAGTGAGGTTCCGgttcccat_ (KDR) (SEQ ID NO: 44) 19136tgAGGAAGGAGGACGAAGGCctctacac_ (KDR) (SEQ ID NO: 45) 19119atGATGACGCCCAGGAGcttcaccaccc_ (RIPK1, (SEQ ID NO: 46) pair A) 19120gaGGAAGGGAAGTActccctggtgatgg_ (RIPK1, (SEQ ID NO: 47) pair A) 19123gtGCAGTGAACCAGGCTGTTctgtggct_ (RIPK1, (SEQ ID NO: 48) pair B) 19124gtTCCCAGgGACTTGGGAtgggtcctgt_ (RIPK1, (SEQ ID NO: 49) pair B) 19121aaGGAGGCAAGGCCGAGgtctgcgatct_ (RIPK1, (SEQ ID NO: 50) pair C) 19122aaGATGTGGAGCAAACTGAAtaatgaag_ (RIPK1, (SEQ ID NO: 51) pair C)

Example 2: Genetic Screening for Mutant FokI ZFNs

Using Saccharomyces cerevisiae as a model system, we isolated ZFNmutants displaying a cold-sensitive phenotype with cleavage activitythat is severely diminished at lower temperature but adequate at higherones. Cold-sensitive mutations are particularly interesting becausehistorically they have been shown to occur in genes encoding subunits ofmultimeric protein complexes. These mutations affect protein-proteininteractions predominantly at low temperature. Thus, isolating thisclass of mutants revealed non-null mutations that identify importantresidues within the dimerization interface.

Single-stranded annealing (SSA)-reporter strain and mutant libraryconstruction was performed as follows. Random mutagenesis of the FokInuclease domain was done using error-prone PCR and the library ofmutants was constructed by gap repair in Saccharomyces cerevisiae.Briefly, the reporter strain was co-transformed with the mutagenized PCRfragment (FokI domain) and a linearized plasmid vector prepared suchthat the ends of the vector shared DNA sequence with the ends of the PCRfragment. Homologous recombination between the vector and the PCRfragment occurred at a high frequency and resulted in a collection ofyeast transformants, containing a mutated ZFN expression vector. Thezinc finger domain of the nuclease binds to the human CCR-5 gene(designated 8266) and is described in detail in U.S. Patent PublicationNo. 2008/0159996.

The library was then screened or selected for phenotypes of interest inbudding yeast, essentially as described in U.S. Patent Publication No.2009/0111119. Briefly, two independent SSA reporter constructs wereintegrated in the genome of budding yeast. Both reporters contain abinding site for a homodimer of the 8266 ZFN. The MEL1 SSA reportercontains both positive and negative selection markers. The URA3 gene isused for positive selection in ura-media and for negative selectionusing 5-Fluoroorotic Acid (5-FOA). The KanMX cassette confers dominantresistance to geneticin (G418). Reconstitution of the MEL1 genefollowing SSA was detected using chromogenic substrates [p-Nitrophenylα-D-galactopyranoside (PNPG) or5-Bromo-4-chloro-3-indoxyl-alpha-D-galactopyranoside (X-a-Gal)]. ThePHOS SSA reporter contains the positive selection cassette NatMXconferring dominant resistance to nourseothricin (NAT) andreconstitution of the PHOS gene was detected using chromogenicsubstrates [p-Nitrophenyl phosphate disodium (PNPP) or x-phosphatep-toluidine salt (X-Phos)]. Therefore, a DNA double-stranded break (DSB)induced by a functional 8266 ZFN induced SSA resulted in reconstitutionof the reporter genes and elimination of positive and negative selectionmarkers.

The genetic screen for FokI mutants was conducted as follows. First,galactose-inducible expression of the ZFNs was performed at thenon-permissive temperature of 22° C. Following recovery, the cells wereincubated in Kan (G418), NAT and ura-media to eliminate all active ZFNs.This step selected for potential cold-sensitive mutants as well as forinactive ZFN.

Second, the cells were shifted to 37° C. (permissive temperature) andplated on media containing 5-FOA and X-Phos. Only cells containing acold-sensitive ZFN formed blue colonies. The plasmids from these cellswere then isolated and retransformed into the reporter strain to confirmthe cold-sensitive phenotype. The resulting mutations were identified bydirect sequencing of the FokI domain.

Table 3 shows various mutants identified by the screen. Mutationspredicted to confer cold sensitivity are indicated in the first column(based on proximity to the dimer interface in ZFNs).

TABLE 3 ZFN cold-sensitive mutants Mutations # isolates I499T 1 I538F 1I538T 3 Q486L 2 Q486L K448M 4 N496D E484V 3 H537L A482T K559T L563M 1Q531R 2 Q531R V512M 1 N500S K402R K427M N578S 1 N500S K469M 1 N476D 1N476K 1 G474S 4 G474A 5 D467E 1

Activity (relative to wild-type) of the cold sensitive cleavageactivities of the isolated mutants is shown in FIG. 1A. The reporterstrain was transformed with the isolated mutant vectors, divided intothree cultures, and incubated at 22° C., 30° C., and 37° C. Followingexpression, the activity of the mutants was determined and reported as afraction of the activity of the wild-type ZFN. The increase in ZFNcleavage activity correlated with elevated temperature of incubationindicates that the isolated mutants are cold-sensitive.

Example 3: Design of Novel Engineered FokI Cleavage Half-Domains

Using the ZFN structure model described in Miller, et al., (2007) Nat.Biotech. 25(7):778-85, we mapped the position of the mutations tested inExample 2 and found out that two of the mutated residues (N496 and H537)face each other on the dimer interface and are found in close proximity.Modelization of those mutations also showed that H537R and N496Dmutations would likely form salt-bridges and strengthen the dimerizationinterface. Table 4 shows the nomenclature of various mutants tested.

TABLE 4 Engineered Cleavage Domain Nomenclature Cleavage domainMutations designation (wildtype residue-position-mutant residue) ELQ486E + I499L ELD Q486E + I499L + N496D ELE Q486E + I499L + N496E KKE490K + I538K KKK E490K + I538K + H537K KKR E490K + I538K + H537R RELH537R + Q486E + I499L DKK N496D + E490K + I538K DD R487D + N496D DADR487D + N496D + I499A RR D483R + H537R RVR D483R + H537R + 538V KIK*E490K + H537K KIR* E490K + H537R DA** R487D + I499A EA** Q486E + I499AKV** E490K + I538V RV** D483R + I538V Sharkey***  S418P + K441EEL-Sharkey Q486E + I499L + S418P + K441E KK-Sharkey E490K + I538K +S418P + K441E ELD-Sharkey Q486E + I499L + N496D + S418F + K441EKKK-Sharkey E490K + I538K + H537K + 8418P + K441E KKR-Sharkey E490K +I538K + H537R + S418P + K441E DA-Sharkey R487D + I499A + S418P + K441EEA-Sharkey Q486E + I499A + S418P + K441E *Note: For the KIK and KIRmutants, the amino acid at position 538 is an isoleucine as is the wildtype. The nomenclature for KIK and KIR uses the ‘I’to distinguish thesemutants from the KK mutants. **Described in Szczepek et al., (2007)Nature Biotechnology 25 (7) p. 786-93. ***Described in Guo et al,, ibid

Various pairwise combinations of the triple mutants (e.g., ELD:KKK,ELD:KKR, ELE:KKK and ELE:KKR) were compared for cleavage activityagainst EL:KK pairs (EL:KK mutants are described in U.S. PatentPublication No. 2008/0131962) in a variety of ZFN backgrounds. TheZFN-containing plasmids were then nucleofected into K562 or PMBC cells.To determine the ZFN activity at the appropriate locus, Cel-1 mismatchassays were performed essentially as per the manufacturer's instructions(Trangenomic SURVEYOR™). Cells were harvested and chromosomal DNAprepared using a Quickextract™ Kit according to manufacturer'sdirections (Epicentre@). The appropriate region of the targeted locuswas PCR amplified using Accuprime™ High-fidelity DNA polymerase(Invitrogen). PCR reactions were heated to 94° C., and gradually cooledto room temperature. Approximately 200 ng of the annealed DNA was mixedwith 0.33 μL Cel-1 enzyme and incubated for 20 minutes at 42° C.Reaction products were analyzed by polyacrylamide gel electrophoresis inIX Tris-borate-EDTA buffer.

As shown in FIGS. 2 to 5, the various combinations of the triple mutantsare more active than the original obligate heterodimeric ZFN (EL:KK). Inparticular, FIG. 2 shows Cel-1 assay results of ZFN variants ELD:KKK,ELD:KKR, ELE:KKK, ELE:KKR, ELD:KIK, ELD:KIR, ELE:KIK and ELE:KIR in ZFNstargeted to 53BP1 3 and 10 days post-transfection of the ZFNs into K562cells. FIG. 3 shows Cel-1 assay results of ZFN variants ELD:KKK andELD:KKR in ZFNs targeted to KDR and 20 days post-transfection of theZFNs into K562 cells. FIG. 4 shows Cel-1 assay results of the ELD; KKRand ELD:KKK FokI engineered cleavage domains in the context ofGR-specific ZFNs in K562 cells. FIG. 4 also shows the cleavageactivities using decreasing amounts of expression plasmid fortransfection (from 400 ng to 16 ng), with two different sets of samples(lanes 1-14 and lanes 15-26). These results show that at 80 ng of inputexpression plasmid, the ELD:KKR and ELF:KKK mutants were both moreactive that the EL:KK mutants (compare lane 8 with lanes 9 and 10 andlane 20 with lanes 21 and 22).

FIG. 5 shows the cleavage activities of the ELD:KKR and ELD:KKK mutantsin three different RIPK1-specific ZFN backgrounds. The new mutants wereboth more active that the EL:KK mutant in RIPK1 pair A and RIPK1 pair B.In FIG. 5B, the new mutants in the pair C background were tested at both37° C. and 30° C. where activity of all ZFP pairs was found to beincreased at 30° C. (see U.S. Patent Publication No: 2009/0111119).

Example 4: Activity of Engineered Cleavage Domains as Homodimers

The new mutants were also tested for their ability to actively cleaveDNA as forced homodimers. In these assays, the zinc finger bindingdomains are fused to a FokI cleavage domain that is the same in bothmembers of the pair. Thus, in order to observe any activity, the FokIdomain must homodimerize with itself (“forced homodimerization”). Forcedhomodimerization of CCR5-targeting ZFNs was assayed by nucleofection ofFokI variants in K562 cells (see FIG. 6) and the Cel-1 assay was used todetermine the frequency of ZFN-induced indels at the CCR5 heterodimertarget, a CCR5-L ZFN homodimer (ABLIM2), and a CCR5-R homodimer (PGC)off-target sites. For these experiments, the mutations were made in theCCR5-specific 8266 and 8196z pair and then tested. Thus, in the laneslabeled “WT”, the 8266/8196z pair was used. Then for each mutant pairtested, a similar pair was made with the indicated mutations, so theEL:EL lanes indicate a pair containing 8266-EL and 8196z-EL and so on.

As can be seen from FIG. 6, whereas the KK, KKK, and KKR homodimers showno detectable cleavage activity at the CCR5 heterodimer target site,there is limited cutting by the EL:EL and ELD:ELD homodimers. TheELD:ELD variants have an approximately 1.5 fold lower activity ascompared to EL:EL indicating increased specificity. Importantly,examination of the known off-target sites ABLIM2 and PGC show nodetectable cleavage activity by any of the mutants.

In order to further confirm the improvement in specificity of the ELDcleavage domain, these same forced homodimers were tested in decreasingconcentrations in K562 cells. As can be seen from FIG. 7, at all DNAconcentration tested, ELD:ELD displays a lower homodimer activity ascompared to EL:EL. Forced GR-specific ZFN homodimers were also tested(see FIG. 8) and there was no cleavage activity detectable. In someembodiments, the I499A mutant was used to replace the I449L mutation tofurther decrease any potential ELD homodimerization. In this case,forced homodimerization of the EAD CCR5 specific ZFN gave no detectablecleavage activity.

In addition, these cells were also tested for DSBs using an antibodyspecific for γ-H2AX which accumulates at DSB sites in the genome. Thestained cells were sorted by flow cytometry, and the results are shownin FIG. 9. As can be seen from the figure, there is very little stainingexcept for the WT pair, indicating a low level of DSBs in the genome inthe presence of the ZFN pairs containing the mutated FokI domains.

Example 5: Activity of the Engineered Half Domains in Primary Cells

The constructs were also tested in primary cells. Decreasing amounts ofCCR5-targeting ZFN constructs containing the indicated mutations werenucleofected in PBMCs, as described in Perez, et al., ibid. The cellswere harvested three days post-transfection and the Cel-1 assay was usedto determine the frequency of ZFN-induced indels. As can be seen fromFIG. 10A, the ELD:KKK and ELD:KKR mutants were quite active in thesecells even at lower concentrations. Similar studies were done with ZFNstargeted to PD-1 and are presented in FIG. 10B. Engineered half domainconstructs were made in three pairs of PD-1-specific ZFNs where pair Acomprised pair 12942 and 12974, pair B comprised pair 12942 and 25016,while pair C comprised pair 12942 and 25029 (see U.S. ProvisionalApplication No. 61/281,432). The results are presented in FIG. 10B in agraphical format and demonstrate that the ELD:KKR and ELD:KKK mutantshad superior activity as compared to both the WT pairs and the EL:KKpairs.

Mutants made in the GR-specific ZFN background were also tested in PBMCsfor activity as shown in FIG. 11A. In this example, decreasing amountsof the mutants were tested at days 3 and 10 post-transfection. The newmutants were found to have increased activity as compared to the EL:KKpair. In FIG. 11B, the mean values of experiments that were repeated insix independent transfections in PBMC are presented. The values aremean+/−standard error of the mean of the relative activities as comparedto the EL-KK pair. P-values use the two-sample T-test and demonstratethe reproducibility of these results.

Example 6: Targeted Integration into the DSB, Comparison of EL:KK andELD:KKR

The EL:KK and ELD:KKR FokI mutant ZFNs were also compared for their usein promoting targeted integration (TI). For this experiment, a donornucleic acid was made containing a novel BamHI restriction site.Following successful TI, the region surrounding the ZFN target site wasamplified via PCR, and then the PCR product was subjected to BamHIrestriction to cleave the newly introduced restriction site. Thesequence of the donor DNA is shown below:

TI Donor DNA: (SEQ ID NO: 52)ggaagttaaagcccatgtttctaatacaatgaacattatgttatgcccaaacttaacaccatcatttcatatgatagcactttcttatagtgttaccttatgctccctgaccaaactcccagacatcaacttgtacttttctattttattctagatctttttgtattgttgttttaaatactttcctgcccattagaggacctaggagccaccctcctctcccctcttaactgatatttagcctttcatgggctttgcatataatggaaatttcaaaatccaccctgagaaatgaaaaccaagtagaggaaaaataaactcttcaaaacacacactaccttccactgctcttttgaagaaaactttacagcttccacaagttaagactccataatgacatcctgaagcttcatcagagcacaccaggcagagtttgggagGTGGTCCTGTTGttgaggcatccagtccagacgggatccagccatactcactgctGTTGAGGAGCTggatggaggagagcttacatctggtctcatgctggggctaaagaaggggaagaacagtgttatgatttaactgtcaaaggaatatcaaaatacagttctcttagcttctcacttcatagtcagaatgctcacagtgaactctggcttcaagtgctagcaggcactaaaatatcctagctaaatatattcaaatcatgttatattcttctttaaacaaaattaagaatgaggtcatttcttttgaagtgtctccaaaatagaatggtgtggttctggttcacttcttcttctttttttttttttttttagatgcttaggatttatttttataatcacg

In this sequence, the ZFN binding sites are shown in capital letters,and the introduced BamHI restriction site is underlined. For theseexperiments, the FokI mutants were tested in the GR-specific ZFNbackground, and as shown in FIG. 11C, were done using two differentZFN-encoding plasmid concentrations during the nucleofection step. Ascan be seen in FIG. 11C, the ELD/KKR pair was more efficient atresulting in the introduction of the donor than the EL/KK pair, at bothconcentrations tested.

Example 7: Comparison of the Activity of DA:RV FokI Mutants VersusELD:KKR or ELD:KKK

ZFN pairs were constructed containing the FokI mutations in both theGR-specific and CCR5-specific ZFN backgrounds. These were then testedagainst their endogenous targets in K562 cells as described above, andassayed for cleavage activity using the Cel-1 mismatch assay asdescribed above. In each set of experiments, 80 ng of DNA encoding theZFNs was used in the nucleofection step. At day 3 followingtransduction, the Cel-1 assays were performed and the results are shownin FIG. 12, which shows the results for the GR-specific andCCR5-specific cleavage. The data demonstrates that the DA:RV FokI pairdisplayed much less activity than the EL:KK, ELD:KKK and ELD:KKR pairs.The EA:KV pair however showed activity in this assay.

Next, the various ZFNs were tested for their ability to homodimerize byforced homodimerization (see Example 4). Typically, it is undesirablefor two FokI mutant domains to have the ability to homodimerize becausethis may increase the potential for unwanted off target cleavage. Theexperiments were carried out as above except that 400 ng of ZFNcontaining plasmid were used for each nucleofection. The results areshown in FIG. 13 and demonstrate that the KV FokI mutant has the abilityto homodimerize to a significant extent. Thus, although the EA:KV pairwas found to have activity comparable to the ELD:KKR pair in this Cel-Iassay (see FIG. 12), the fact that the KV FokI mutant was able tohomodimerize and display cleavage activity makes it less desirablebecause of the increased risk of off-site cleavage.

Example 8: Enhancement of the Activity of the DA:RV FokI Mutants

The DA:RV FokI mutants were then examined to see if it would be possibleto increase their activity by combining them with other FokI mutations.Thus, the DA:RV pairs were made to include the N496D and H537R mutationsresulting in a DAD:RVR pair. The Cel-I activity assay results for theCCR5-specific and CXCR4-specific pairs including these mutants are shownin FIG. 14. Experiments were carried out as previously described using80 ng of plasmid per transduction. As can be seen from the Figure, theaddition of the N496D and H537R mutations increased cleavage activity.Similar results were also found using these mutations in the GR-specificZFN background (FIG. 14).

The DA:RV+N496D and H537R combination was also tested in a less activeZFN pair background. In this experiment, KDR-specific ZFNs were chosenand the results of the Cel-I assay are shown in FIG. 15. In this figure,the activity of both the DA:RV and DAD:RVR mutants were not detectable.Thus, the addition of the N496D and H537R mutations is helpful in someZFNs but is not able to rescue ZFN pairs that have weak or undetectableactivity.

Example 9: Testing of Orthogonal Pairs for Simultaneous Specific DualCleavage

It may be desirable to perform simultaneous cleavage at two target siteswithin a genome. For added specificity, it would be best if only the ZFNpairs that cleave at the desired locus are able to productively dimerizesuch that an active pair has the specificity desired. To achieve thisgoal, pairs must not be able to homodimerize or transheterodimerize tocreate an active pair. In other words, if target 1 is cleaved by ZFNpair A+B, and target 2 is cleaved by ZFN pair X+Y, pairings of A+A(homodimers), A+X and A+Y (transheterodimers), for example, would beundesirable. Thus, the ELD/KKR+DAD/RVR pairs specific for CCR5 and CXCR4were tested together with the hopes that the CCR5-specific ELD halfcleavage domain would not be able to transheterodimerize with either theCXCR5-specific DAD or CXCR4-specific RVR half domains. In addition,variants of the ELD/KKR pair were made such that the D mutation atposition 496 in the ELD mutant and the R mutation at position 537 in KKRwere exchanged to form a REL/DKK pair(H537R+Q486E+I499L/N496D+E490K+I538K). In addition, the ELD/KKR+DD/RRpairs specific for CCR5 and CXCR4 were also tested together.

The Cel-I activity assay results are shown in FIGS. 16 and 22. In thisexperiment, the conditions tested were both a standard 37° C. incubationas well as a 30° C. incubation (see co-owned U.S. Pat. No. 8,772,008).Briefly, following transduction, the cells were held at 37° C. for 3days or at 30° C. for 3 days. Following incubation for 3 days, Cel-Iassays were performed to see if both targets were cleaved, where theCCR5-specific Cel-I assay is shown on the top in FIG. 16, and theCXCR4-specific Cel-1 assay is shown below.

These results indicate that cleavage at both the CCR5 and CXCR4 targetswas achievable in a single step using these pairs of orthogonal mutants.

The mutants were further tested to examine potential off targetcleavage. An in silico analysis was done to identify potential offtarget sites that might resemble a target that could be recognized by anillegitimate CCR5-CXCR4 transheterodimer ZFN pair. In these experiments,the four top candidates for off target cleavage were examined by theCel-I assay, where the sequences for the off-target sites are listedbelow in Table 5.

TABLE 5 Potential Off-Target Sites La- bel Pair Locus Target Sequence* #3 CCR5-L C1orf210 CaTCATCCTCATCTTCAGCcACCTGTGGGcGG CXCR4-(SEQ ID NO: 53) R  #5 CCR5-L TBC1D5 GGcaATaCTCATCTTCACTGACCTGaGGGTGGCXCR4- (SEQ ID NO: 54) R  #7 CCR5-R CD274ACtCCcCTaCTtCAACATAAACTGCAAAAGG CXCR4- (SEQ ID NO: 55) L #10 CCR5-R FRYLAGACacCTTCaACTGCTTAAACTGaAAAAGG CXCR4- (SEQ ID NO: 56) L *Capitalletters in the target sequence indicate potential contact points, lowercase letters indicate nucleotides in the sequence that are not thoughtto contact the ZFP.

The transductions were tested as above using both the 37° C. and 30° C.incubation conditions, and the results are shown in FIGS. 17 and 22. Asshown in FIG. 17, the ELD/KKR CCR5 pair in combination with the ELD/KKRCXCR4 pair gave some cleavage at off targets #3, #5 and #10. The ELD/KKRCCR5 pair combination with the REL/DKK CXCR4 pair also gave somecleavage at sites #3, #5 and #10. However, the ELD/KKR CCR5 pair,combined with the DAD/RVR CXCR4 pair gave no detectable cleavage atthese off target sites. Furthermore, as shown in FIG. 22, the ELD/KKRCCR5 pair, combined with the DD/RR CXCR4 pair gave no detectablecleavage at these off target sites.

These results demonstrate that these FokI mutants may be used in sets toallow for simultaneous cleavage of more than one target site at a time,while decreasing undesirable off target cleavage.

Example 10: Evaluation of FokI Mutants Paired with the Sharkey Mutant

A set of FokI mutants have been described which are thought to enhanceefficiency of DNA cleavage (see Guo, et al., ibid), which are known asthe Sharkey (S418P+K441E) and Sharkey′(S418P+F432L+K441E+Q481H+H523Y+N527D+K559Q) FokI mutants. Thus, theSharkey mutant was tested in combination with the various FokI mutantsdescribed herein to see if cleavage activity could be further enhancedby the presence of the Sharkey mutations. The mutant combinations weremade in the GR-specific and the KDR-specific ZFN backgrounds and testedfor cleavage activity using the Cel-I assay as described above. Theresults are shown in FIG. 18, and demonstrate that the activities of themutations appear to be additive. For example, comparison of lanes 10 and11 with lanes 12 and 13 on the day 3 panel shows that the detected NHEJactivity (indels) went from 11-12 for the ELD/KKR GR-specific pair to20% indels for the ELD-S/KKR-S pair. Similarly, comparison of lanes 10and 11 with lanes 12 and 13 for the KDR specific ZFNs at day 3 went fromapproximately 26-28% indels detectable to 48-50% indels detectable.

In addition, Sharkey FokI mutants were also combined with the DA/RV andDAD/RVR FokI mutants in the GR-specific ZFN background, and tested foractivity using the Cel-I activity assay. The results are presented inFIG. 19A and show that the FokI mutations are additive in terms ofactivity. (Compare lanes 4 and 5 with lanes 6 and 7).

The mutant combinations were also tested to see if the presence of theSharkey mutation increased the amount of homodimerization cleavage in aforced homodimerization assay as described above in Example 4. FIG. 20shows the results of the GR-specific mutants, with or without the addedSharkey mutation. As can be seen from the figure, the mutants were notdetectably altered in the homodimerization capability. Similarly, theDA-S/RV-S and DAD-S/RVR-S mutants were also tested to see if there wasan increase in homodimerization in the GR-specific ZFN background. Theresults are shown in FIG. 21A, and demonstrate that there was not anincrease in productive homodimerization. FIG. 21B demonstrates an equalloading of all lanes.

Example 11: Enhancement of the Activity of the D:R FokI Mutants

The D:R FokI mutants (R487D:D483R) (see, e.g., U.S. Patent PublicationNos. 2008/0131962 and 2009/0305346) were examined to see if it would bepossible to increase their activity by combining them with other FokImutations. Briefly, the D:R pairs were made to include the N496D andH537R mutations resulting in a DD:RR pair and Cel-I assays performed asdescribed above.

As shown in FIG. 21, the addition of the N496D and H537R mutationsincreased cleavage activity.

In sum, these results demonstrate that FokI mutants described herein arethe data presented here demonstrate that the novel mutants are moreactive and display less off-site cleavage activity than the previouslydescribed FokI mutants.

What is claimed is:
 1. An artificial nuclease comprising: (i) aDNA-binding domain that binds to a target site in the genome of thecell; and (ii) a polypeptide comprising an engineered FokI cleavagehalf-domain consisting of substitution mutations numbered relative to awild-type FokI sequence as shown in SEQ ID NO:57, wherein thesubstitution mutations are as follows: the wild-type isoleucine (I)residue at position 499 is replaced with a threonine residue (T)(I499T); the wild-type 1 residue at position 538 is replaced with aphenylalanine (F) residue (I538F); the wild-type 1 residue at position538 is replaced with a T residue (I538T); the wild-type Q residue atposition 486 is replaced with a L residue and the wild-type lysine (K)residue at position 448 is replaced with a methionine (M) residue(Q486L:K448M); the wild-type asparagine (N) residue at position 496 isreplaced with an aspartic acid (D) residue and the wild-type glutamicacid (E) residue at position 484 is replaced with a valine (V) residue(N496D:E484V); the wild-type histidine residue at position 537 isreplaced with a L residue, the wild-type alanine (A) residue a position482 is replaced with a T residue, the wild-type lysine (K) residue atposition 559 is replaced with a T residue and the L residue at position563 is replaced with an M residue (H537L:A482T:K559T:L563M); thewild-type Q residue at position 531 is replaced with an arginine (R)residue (Q531R); the wild-type Q residue at position 531 is replacedwith a R residue and the wild-type V residue at position 512 is replacedwith a M residue (Q531R:V512M); the wild-type N residue at position 500is replaced with a serine (S) residue, the wild-type K residue atposition 402 is replaced with an R residue, the wild-type K residue atposition 427 is replaced with an M residue and the wild-type N residueat position 578 is replaced with a S residue (N500S:K402R:K427M:N578S);the wild-type N residue at position 500 is replaced with a S residue andthe wild-type K residue at position 469 is replaced with an M residue(N500S:K469M); the wild-type N residue at position 476 is replaced witha D residue (N476D) or a K residue (N476K); the wild-type glycine (G)residue a position 474 is replaced with a S residue (G474 S); thewild-type G residue at position 474 is replaced with an A residue(G474A); or the wild-type D residue at position 467 is replaced with anE residue (D467E).
 2. The artificial nuclease of claim 1, furthercomprising an additional amino acid substitution at one or more ofpositions 418, 432, 441, 481, 483, 486, 487, 490, 496, 499, 523, 527,537, 538 and 559, numbered relative to a wild-type FokI sequence asshown in SEQ ID NO:57.
 3. An isolated cell or cell line comprising cellscomprising the artificial nuclease of claim
 1. 4. The isolated cell orcell line of claim 3, wherein the cells comprise a genomic modification.5. The isolated cell or cell line of claim 4, wherein the genomicmodification comprises a deletion.
 6. The isolated cell or cell line ofclaim 4, wherein the genomic modification comprises an insertion.
 7. Theisolated cell or cell line of claim 6, wherein a donor moleculecomprising a sequence encoding a polypeptide is inserted into thegenome.
 8. The isolated cell or cell line of claim 7, wherein a donormolecule comprising a sequence encoding an RNA is inserted into thegenome.
 9. A method of making an isolated cell comprising a genomicmodification, the method comprising simultaneously cleaving the genomeof the cell using first and second artificial nucleases according toclaim 1 such that the cleaved genome is genetically modified.